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IDT offers several free online tools for qPCR primer and probe design. The PrimeTime™ Predesigned qPCR Assays Design Tool, PrimerQuest™ Tool, OligoAnalyzer™ Tool, and RealTime PCR Tool guide you through identifying primer and probe sequences to make sure they are specific to your assay. Because these tools contain design engines that use sophisticated algorithms that, for example, take into account nearest neighbor analysis to calculate Tm, they generally provide the very best designs.
Keep in mind: probes used in qPCR should have a melting temperature at least 6 degrees higher than the flanking primers and should be 28 bases or shorter. Probe designs should be screened for self-dimers and heterodimers against the primers, as well as hairpins. Also, run a BLAST™ alignment to ensure probe efficiency will not be reduced due to off target interactions.
For more information review IDT's primer design tools for PCR and qPCR or contact us for help with custom designs.
Also read our DECODED article, How to design primers and probes for PCR and qPCR.
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